1. Field of the Invention
The present invention relates to compounds that affect the fidelity of eukaryotic translation initiation codon selection and screening methods for identifying such compounds.
2. Background of the Invention
Translation of mRNA into functional protein is energetically expensive and must be highly accurate. The initiation phase of protein synthesis establishes the reading frame of translation and commits the cellular machinery to begin the elongation phase. For most mRNAs, the start codon is an AUG. It has been shown that codons that vary from AUG in one position (near-cognates) can be used up to ˜8% as efficiently as AUG codons as start sites in S. cerevisiae (Zitomer et al., Mol. Cell. Biol., vol. 4, no. 7, pp. 1191-1197, 1984; Clements et al., Mol. Cell. Biol., vol. 8, no. 10, pp. 4533-4536, 1988; Donahue et al., Mol. Cell. Biol., vol. 8, no. 7, pp. 2955-2963, 1988; Kolitz et al., RNA, vol. 15, no. 1, pp. 138-152, 2009). Translation initiation at non-AUG codons has been shown to occur naturally in both mammals and yeast. For example, CAPC, a protein over-expressed in some cancers, initiates translation using a non-AUG start codon (Anaganti et al. 2009). In yeast, two tRNA synthetase genes, GRS1 and ALA1, use non-AUG start codons for normal expression (UUG and ACG, respectively) (Chang and Wang 2004; Tang et al. 2004). Additionally, using ribosomal profiling Ingolia et al. (2009) identified 143 actively translated upstream open reading frames (uORFs) that appear to have non-AUG start codons in yeast. Interestingly, translation from these small uORFs is increased upon amino acid starvation, although neither the reason for this effect nor its mechanism is yet understood (Ingolia et al. 2009).
Recent studies using a reconstituted S. cerevisiae translation initiation system have elucidated core events involved in start codon selection. Briefly, the 40S subunit with eukaryotic initiation factor (eIF) 1, eIF1A, the ternary complex (TC: eIF2, initiator methionyl-tRNA and GTP) and eIF5 (the GTPase activating protein (GAP) for eIF2) is loaded onto the 5′ end of the mRNA and scans to locate the start codon. In vivo, eIF4F, eIF4B and eIF3 are involved in loading of this 43S ribosomal pre-initiation complex (PIC) onto the 5′ end of the mRNA and subsequent scanning of the message. After the initiator tRNA anti-codon base pairs with the mRNA start codon, eIF1 is released from the complex. Loss of eIF1 in turn allows inorganic phosphate to be released from eIF2, converting the factor into its GDP-bound form. The release of eIF1 also produces a conformational change in the complex that is thought to prevent further scanning. At this stage, the large ribosomal subunit joins the small ribosomal complex with the help of eIF1A and eIF5B, producing an 80S initiation complex that can enter the elongation phase of the cycle (for reviews of the mechanism of eukaryotic translation initiation see (Lorsch et al., J. Biol. Chem., vol. 285, no. 21203-21207, 2010; Sonenberg et al., Cell, vol. 136, no. 4, pp. 731-745, 2009; (Jackson et al., Nat. Rev. Mol. Cell. Biol., vol. 11, no. 2, pp. 113-127, 2010).
Although many steps involved in locating the start codon have been elucidated, the mechanistic details of this process are still a mystery. Many components of the translation machinery are known to impact the fidelity of start codon selection. Mutations in eIF1 (Yoon et al., Mol. Cell. Biol., vol. 12, no. 1, pp. 248-260, 1992), 1A (Fekete et al., EMBO J., vol. 24, no. 20, pp. 3588-3601, 2005; Saini et al., Genes Dev., vol. 24, no. 1, pp. 97-110, 2010), eIF2 (Donahue et al., Cell, vol. 54, no. 5, pp. 621-632, 1988; Castilho-Valavicius et al., Genetics, vol. 124, no. 3, pp. 483-495, 1988), eIF5 (Huang et al., Genes Dev., vol. 11, no. 18, pp. 2396-2413, 1997), eIF3 (Valasek et al., Mol. Cell. Biol., vol. 24, no. 21, pp. 9437-9455; 2004) and eIF4G (He et al., Mol. Cell. Biol., vol. 23, no. 15, pp. 5431-5445, 2003) are known to decrease the fidelity of start codon selection in vivo (Sui− phenotype), and have been important tools to study the steps involved in translation initiation. While mechanistically very different, the selection of the start codon in the P-site during translation initiation can be related to selection of tRNA in the A-site during elongation; both processes are dependent on matching codon:anti-codon base pairing, which triggers downstream events (Kolitz et al., RNA, vol. 15, no. 1, pp. 138-152, 2009; Cigan et al., Science, vol. 242, no. 4875, pp. 93-97, 1988; Ogle et al., Science, vol. 292, no. 5518, pp. 897-902, 2001). Small molecules such as the aminoglycoside family of antibiotics have been crucial tools to probe the mechanism of tRNA selection in the ribosomal A-site during the elongation phase of translation (Rodnina et al., Annu. Rev. Biochem., vol. 70, pp. 415-435, 2001; Ogle et al., Cell, vol. 111, no. 5, pp. 721-732, 2002; Ogle et al., Annu. Rev. Biochem., vol. 74, pp. 129-177, 2005). Although mutations in eukaryotic initiation factors have been studied, no chemical modulators of start codon selection exist to help elucidate the mechanism of this complicated process. Compounds that increase or decrease misreading during initiation could provide unique insight to how AUG is selected during initiation.